Genetic test for the identification of carriers of complex vertebral malformations in cattle

ABSTRACT

Genetic markets for identifying bovine carriers of complex vertebral malformation (CVM) disease gene are described. The genetic markers, including the microsatellite markers BM4129, INRAA003, BMS2790, ILSTS029, INRA123, BM220, HUJ246, BMS862, BMS937, BL1048, BMS2095 and BMS1266 and the bovine SLC35A3 gene, are located on bovine chromosome BTA3. The G/T polymorphism at position 559 of the bovine SLC35A3 gene is identified as being causative and diagnostic for CVM in cattle.

FIELD OF THE INVENTION

The present invention relates generally to a genetic disease observed inbovines termed Complex Vertebral Malformation (CVM). More particularly,the invention relates to molecular markers for identifying potentialbovine carriers of CVM and for identifying the CVM gene locus andmutations thereof responsible for complex vertebral malformation inbovines.

BACKGROUND OF THE INVENTION

Complex Vertebral Malformation (CVM) is a congenital vertebral disorderdetected in Holstein-Friesian (HF) black and white dairy cattle. Thedisease has recently been described (Agerholm et al., 2000). In Denmark,all cases diagnosed until today (Oct. 17, 2000) have been geneticallyrelated to the former elite US Holstein bull Carlin-M Ivanhoe Bell.According to the present data, CVM appears to be inherited as anautosomal recessive disease.

The disease is characterised by a congenital bilateral symmetricarthrogryposis of the distal joints and malformations of the columna,mainly at the cervico-thoracic junction combined with reduced bodyweight (Agerholm et al., 1994).

Externally, there are the following major findings: In many cases thecervical and/or the thoracic part of the columna seems to be short.Moderate bilateral symmetric contraction of the carpal joints and severecontraction and supination of the phalango-metacarpal joint (fetlock)are constant findings. Contraction and pronation of thephalango-metatarsal joint and slight extension of the tarsus are alsocommon findings. In most cases an irregular course of the columna aroundthe cervico-thoracic junction is observed. Scollosis may be observed,and lesions may be present in other regions of the columna. Theirregular course is often recognised by inspection and palpation of theventral aspect of the columna. However, lesions may be minimal andrestricted to two or few vertebrae. In such cases the columna may be ofalmost normal length. Therefore, radiological examination of the columnais recommended to exclude vertebral malformations in suspected cases.The spinal cord is of normal size lying with the vertebral canal withoutobvious compressions. Using radiology, complex vertebral malformationsconsisting of hemivertebrae, fused and malshaped vertebrae, scollosis,and anchylosis are found at varying degrees. This is best demonstratedfollowing removal of the arcus vertebrae. In some cases malformations ofthe heart are present, mostly as a high interventricular septal defectand eccentric hypertrophy of the right ventricle. Malformations of thelarge vessels may occur. In the lungs fetal atelectasis is present.Serohemorrhagic fluids are most present in the thoracic cavity. Avariety of other malformations have been observed, but these are notconstant or common findings. Lesions due to dystocia are often found.

Malformations have been observed both in aborted fetuses, prematurelyborn calves and in stillborn calves born at term. Cases among oldercalves have not yet been observed. In general the body weight isreduced, and the body weight is lower in premature born calves than incalves born at term.

Additionally, there seems to be an increased frequency of abortions incows inseminated with semen of carrier bulls. At present the cause ofthis is unknown.

Presently, the only tool available for CVM diagnosis is patho-anatomicaldiagnosis based on the above described presence of bilateral symmetricarthrogryposis of the distal joints and malformations of the columna,mainly at the cervico-thoracic junction combined with reduced bodyweight. However, symmetric contractions of the limbs are common andgeneral findings in vertebral malformations in calves. Therefore,differential diagnostic problems do exist as it is often difficult todifferentiate between CVM and other malformations.

The fact that the genetic defect appears to be spread by the bullCarlin-M Ivanhoe Bell which has been used intensively all over the worldmakes it of significant economic importance to be able to test whethercurrent and potential breeding bulls are carriers of the defect.

In order to obtain an estimate of the frequency of potential CVM carrieranimals within the Danish cattle population, the present inventors haveextracted pedigree information from the Danish national cattle database.At the time of the extraction (October 2000) there were registered919,916 pure-bred cows and heifers, and 169,821 pure-bred bulls and malecalves. Bell was found 707,915 times in the pedigrees of the cows andheifers and 161,043 times in the male pedigrees. In Tables 1 and 2below, the number of occurrences of Bell in each generation of thepedigrees is shown.

TABLE 1 Occurrence of Bell in the pedigrees of Danish Holstein cows andheifers Generations Cumulative NR Frequency Percent Frequency 2 212403.0 21244 3 202460 28.6 223704 4 321043 45.4 544747 5 133956 18.9 6787036 27307 3.9 706010 7 1869 0.3 707879 8 36 0.0 707915

TABLE 2 Occurrence of Bell in the pedigrees of Danish Holstein bullsCumulative Generation Frequency Percent Frequency 2 436 0.3 436 3 2014412.5 20580 4 82394 51.2 102974 5 44545 27.7 147519 6 12455 7.7 159974 71040 0.6 161014 8 29 0.0 161043

Although these numbers also include some double and triple occurrencesof Bell in the pedigrees, the data dearly show that a majority of theDanish Holstein cattle are potential carriers of CVM. Clearly, theproblem is immense on a global scale.

Thus, there is great demand in the cattle industry for a genetic testthat permits the identification of cattle in various breeds that arepotential carriers of CVM (e.g. before detectable onset of clinicalsymptoms).

Prior to the present invention, microsatellite mapping has not beenapplied to the gene causing the above complex vertebral malformationswhich has not been isolated or characterised. Thus, to the inventors'best knowledge, the diagnostic method according to the inventiondescribed in further detail in the following has not previously beensuggested or disclosed.

Accordingly, the present invention, which comprises mapping of thedisease locus for CVM, has provided a DNA test based on microsatellitemarkers located on bovine chromosome BTA3. The ability of the test todefine the carrier status of animals descending from Bell has beenconfirmed which appears from the examples below.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention provides a method fordetecting the presence in a bovine subject of a genetic markerassociated with bovine complex vertebral malformation (CVM), comprisingthe steps of providing a bovine genetic material, and detecting in thegenetic material the presence or absence of at least one genetic markerthat is linked to a bovine complex vertebral malformation disease traitor a specific nucleotide polymorphism which causes the complex vertebralmalformation disease trait.

In a further aspect, the invention pertains to a diagnostic kit for usein detecting the presence in a bovine subject of at least one geneticmarker associated with bovine complex vertebral malformation (CVM),comprising at least one oligonucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37 and SEQ ID NO: 38, and combinations thereof.Furthermore, the invention relates to a diagnostic method including adiagnostic kit for the detection of a G/T polymorphism in the bovineSLC35A3 gene causative and diagnostic for CVM in cattle.

DETAILED DISCLOSURE OF THE INVENTION

One primary objective of the present invention is to enable theidentification of cattle carrying bovine complex vertebral malformation(CVM). This is achieved by a method which detects the presence of agenetic marker associated with bovine CMV in a bovine subject. Morespecifically, the genetic marker may be the bovine SLC35A3 gene or evenmore specifically specific polymorphisms in the bovine SLC35A3 gene.

As used herein, the term a “bovine subject” refers to cattle of anybreed. Thus, any of the various cow or ox species, whether male orfemale, are included in the term, and both adult and new-born animalsare intended to be covered. The term does not denote a particular age.One example of a bovine subject is a member of the Holstein-Friesiancattle population.

The term “genetic marker” refers to a variable nucleotide sequence(polymorphic) that is present in bovine genomic DNA on a chromosome andwhich is identifiable with specific oligonucleotides. Such a variablenucleotide sequence is e.g. distinguishable by nucleic acidamplification and observation of a difference in size or sequence ofnucleotides due to the polymorphism. In useful embodiments, such geneticmarkers may be identified by several techniques known to those skilledin the art, and include typing of microsatellites or short tandemrepeats (STR), restriction fragment length polymorphisms (RFLP),detection of deletion or insertion sites, and random amplifiedpolymorphic DNA (RAPD) as well as the typing of single nucleotidepolymorphism (SNP) by methods including restriction-fragment-lengthpolymerase chain reaction, allele-specific oligomer hybridisation,oligomer-specific ligation assays, mini-sequencing, direct sequencing,fluorescence-detected 5′-exonuclease assays, and hybridisation with PNAand LNA probes and others. However, it will be appreciated that othergenetic markers and techniques may be applied in accordance with theinvention.

As described above, “bovine complex vertebral malformations” (CVM) is acongenital vertebral disorder. Presently, the disease has only beendetected in Holstein-Friesian (HF) black and white dairy cattle;however, it is also contemplated that other bovine races may beaffected. The disease has recently been described by Agerholm et al.,2000. Accordingly, in the present context CVM and bovine complexvertebral malformation disease trait are to be understood as a diseaseresulting in the clinical symptoms previously described herein, and asreported and defined by Agerholm et al., 2000.

The method according to the invention includes the provision of a bovinegenetic material. Such material include bovine DNA material which may beprovided by any conventional method or means. The bovine DNA materialmay e.g. be extracted, isolated and purified from blood (e.g., fresh orfrozen), tissue samples (e.g., spleen, buccal smears), hair samplescontaining follicular cells and semen.

As previously described, the method of the present invention furthercomprises a step of detecting in the genetic material the presence orabsence of a genetic marker that is linked to a bovine complex vertebralmalformation disease trait.

In order to detect if the genetic marker is present in the geneticmaterial, standard methods well known to persons skilled in the art maybe applied, e.g. by the use of nucleic acid amplification. In order todetermine if the genetic marker is genetically linked to the complexvertebral malformation disease trait, a lod score can be applied. A lodscore, which is also sometimes referred to as Z_(max), indicates theprobability (the logarithm of the ratio of the likelihood) that agenetic marker locus and a specific gene locus are linked at aparticular distance. Lod scores may e.g. be calculated by applying acomputer programme such as the MLINK programme of the LINKAGE package(Lathrop et al., 1985). A lod score of greater than 3.0 is considered tobe significant evidence for linkage between the genetic marker and thecomplex vertebral malformation disease trait or gene locus.

In one embodiment of the invention, the genetic marker is located onbovine chromosome BTA3. The region of bovine chromosome BTA3 comprisingthe genetic markers that are useful in the method of the presentinvention is indicated in FIG. 2.

Accordingly, genetic markers located on bovine chromosome BTA3 in theregion flanked by and including the polymorphic microsatellite markersBM4129 and BMS1266, may be useful according to the present invention. Inone specific embodiment, the at least one genetic marker is located inthe region from about 59.5 cM to about 67.9 cM on bovine chromosomeBTA3.

In a further useful embodiment, the at least one genetic marker islocated on the bovine chromosome BTA3 in the region flanked by andincluding the polymorphic microsatellite markers INRAA003 and BMS937.

In a further aspect, the at least one genetic marker is located on thebovine chromosome BTA3 in the region flanked by and including thepolymorphic microsatellite markers INRAA003 and ILSTS029.

In another advantageous embodiment, the at least one genetic marker isselected from the group consisting of microsatellite markers BM4129,INRAA003, BMS2790, ILSTS029, INRA123, BM220, HUJ246, BMS862, BMS937,BL1048, BMS2095 and BMS1266.

As described in the examples, the at least one genetic marker may belinked to a gene causing the bovine complex vertebral malformationdisease. Thus, in one embodiment, the at least one genetic marker islocated on bovine chromosome BTA3 in the region flanked by and includingthe polymorphic microsatellite markers BM4129 and BMS1266 andgenetically linked to the CVM disease trait or the CVM gene locus at alod score of at least 3.0, such as at least 4.0, including at least 5.0,such as at least 6.0, including at least 7.0 such as at least 8.0,including at least 9.0 such as at least 10.0, including at least 11.0,such as at least 12.0.

The specific definition and locus of the above polymorphicmicrosatellite markers can be found in the USDA genetic map (Kappes etal., 1997).

It will be appreciated that in order to detect the presence or absencein a bovine subject of a genetic marker associated with CVM, more thanone genetic marker may be applied in accordance with the invention.Thus, the at least one marker can be a combination of two or moregenetic markers which are shown to be informative whereby the accuracyof the test can be increased.

Accordingly, as further exemplified below, in one useful embodiment, twoor more of the microsatellite markers INRAA003, BMS2790, ILSTS029, INRA123, BM220, HUJ246, BMS862, BMS937 can be used in combination.

In accordance with the invention, the nucleotide sequences of the primerpairs for amplifying the above microsatellite markers are described inTable 4 below.

The comparative maps (Solinas-Toldo et al., 1995) show that most ofbovine chromosome BTA3 corresponds to a part of human chromosome 1(HSA1). The genetic mapping of the CVM locus presented herein makes itpossible to use the information available about human genes and toconcentrate the search for the candidate gene to genes present on humanchromosome 1. This will greatly limit the number of candidate genes andfacilitate the search for the CVM causative gene.

Genetic markers of the present invention can be made using differentmethodologies known to those skilled in the art. Thus, it will beunderstood that with the knowledge presented herein, the nucleotidesequences of the above described polymorphic microsatellite markers ofbovine chromosome BTA3 have been identified as being genetically linkedto the CVM gene locus, and additional markers may be generated from theknown sequences or the indicated location on bovine chromosome BTA3 foruse in the method of the present invention.

For example, using the map illustrated in FIG. 2, the CVM region ofbovine chromosome BTA3 may be micro-dissected, and fragments cloned intovectors to isolate DNA segments which can be tested for linkage with theCVM gene locus. Alternatively, with the nucleotide sequences provided inTable 4, isolated DNA segments can be obtained from the CVM region bynucleic add amplification (e.g., polymerase chain reaction) or bynucleotide sequencing of the relevant region of bovine chromosome BTA3(“chromosome walking”).

Additionally, the above described homology between bovine chromosomeBTA3 and human chromosome 1 (HSA1) indicates that any gene or expressedsequence tag that is mapped to this analogous region in human may alsomap to the CVM region of bovine chromosome BTA3. Thus, genes orconserved sequences that map on human chromosome HSA1 may be analysedfor linkage to the CVM gene locus using routine methods.

Genotyping is based on the analysis of genomic DNA which can be providedby using standard DNA extraction methods as described herein. When thegenomic DNA is isolated and purified, nucleic add amplification (e.g.polymerase chain reaction) can be used to amplify the region of the DNAcorresponding to each genetic marker to be used in the analysis fordetecting the presence in a bovine subject of a genetic markerassociated with CVM. Accordingly, a diagnostic kit for use in such anembodiment comprises, in a separate packing, at least oneoligonucleotide sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 andSEQ ID NO: 38, and combinations thereof.

Identification of a Mutation in the Bovine SLC35A3 Gene Causative andDiagnostic for CVM in Cattle

Having established the genomic localisation of the CVM gene delimited bypolymorphic microsatellite markers, a search for the identification ofthe structural gene and the causative mutation herein was performedwhich can be used as an ultimate genetic marker for CMV.

The human genome sharing sequence homology to the CVM region, defined asthe region between the markers INRA003 and ILSTS029, was identified. Themarker BMS2790 is located in this interval (FIG. 1). Initially, 5different clones from a bovine BAC library (RPCI-42, constructed andmade available by P. de Jonge and co-workers) were identified, eachharbouring one of the markers INRA003, ISLTS029, or BMS2790. Usingsequence information obtained from these BAC dones, a single region onhuman chromosome 1 which contained a region of homology to themarker-containing BACs was identified using the BLASTN programme onpublic sequence databases. This region spans about 6 million base pairsand is located in position approx. 107.4–113.5 (FIG. 3) (ENSEMBL viewer,The Sanger Centre).

Isolation and Sequencing of the SLC35A3 cDNA

Based on a homology alignment of the SLC35A3 gene between homo sapiensand canis familiaris, 2 oligos (SL1F and SL8R) were designed foramplification of almost the entire cDNA for bovine SLC35A3, includingthe start codon. PCR was performed on cDNA isolated from heart tissuesamples collected from a wildtype animal, a CVM carrier, and an affectedanimal, respectively. To obtain the sequence of the 3′-end of the gene,the resulting PCR fragment was sequenced and a new oligo designed(SL5F). To amplify the 3′ end of SLC35A3, SL5F was used in combinationwith an oligo (bSLCBVIR), designed using the published sequence of apartial bovine EST (genbank, dbEST). The cDNA sequence (SEQ ID NO: 18)and the translated peptide sequence (SEQ ID NO: 17) of bovine SLC35A3 isshown in FIG. 4. The protein encoded by bovine SLC35A3 contains 326amino acids and shares homology to a family of previously known proteinsinvolved in the transport of nucleotide sugars from the cytosol into theGolgi lumen. The alignment depicted in FIG. 5 shows the homology toSLC35A3 proteins previously described in human (Ishida et al. 1999) andin dog (Guillen et al. 1998).

Detection of a Polymorphism in the SLC35A3 Gene

To detect potential polymorphisms in the bovine SLC35A3 gene, PCRamplification of the gene was performed using cDNA isolated from hearttissue samples collected from a CVM carrier and an affected animal,respectively. Sequencing of the fragment isolated from the affectedanimal revealed a sequence identical to the wildtype, except for theaffected animal being homozygous for the nucleotide T in nucleotideposition 559, compared to the wildtype animal being homozygous for G inthe corresponding position (see FIG. 4). Sequencing of the cDNA from ananimal being carrier of the CVM-defect showed this animal to beheterozygous having both T and G in position 559.

The exchange of G to T in position 559 affects the sequence of theresulting peptide in changing a valine in position 180 to aphenylalanine (see FIG. 4).

Typing the SLC35A3 Polymorphism by a DNA Sequencing Based Assay

FIG. 6 shows the results obtained from sequencing a PCR fragmentamplified from genomic DNA and containing the region (from 544 to 572 ofthe SLC35A3 cDNA, for numbering see FIG. 4) containing the G/T mutation.The left and right panels show forward and reverse sequencing,respectively. The upper row (marked by −/−) shows the wildtype result,showing a G in the polymorphic position using forward sequencing and a Cin the similar position on the other strand using reverse sequencing(marked by asterisks). The lower row +/+ shows the results from anaffected calf, showing a T in the polymorphic position using forwardsequencing and an A in the similar position on the other strand usingreverse sequencing (marked by asterisks). The heterozygote (+/−) isshown in the middle panel and expectedly displays as a mixture of thewildtype and affected signal and thus has both a T and a G signal usingforward sequencing and an A and a C signal on the other strand.

All Calves Affected by CVM are Homozygous for the T-allele

Genotyping of 39 calves affected by CVM was performed by sequencing of aPCR product amplified from genomic DNA (see FIG. 6 and example 6). Allof these animals were homozygous for the T-allele, confirming theinitial results.

The T-allele is not Found in Animals Unrelated to Bell

Since calves affected by CVM have been reported only in pedigreescontaining the widely used bull BELL, it was investigated whether theT-allele was present in animals unrelated to BELL. Taking advantage ofthe Danish Cattle Database, 496 animals of the Holstein breed withoutBELL in their pedigree were identified and sampled. Genotyping of theseanimals was performed by sequencing of a PCR product amplified fromgenomic DNA (see FIG. 6 and example 6). None of these animals containedthe T-allele, suggesting that this allele is found exclusively in theline of animals closely related to BELL.

By sequencing, more than 326 unrelated (at least for the last threegenerations) animals of 12 different breed were also genotyped. All ofthese animals were homozygous for the wildtype allele (G-allele) againdemonstrating the lack of the CVM-related allele (the T-allele) in thegeneral cattle population.

Typing the SLC35A3 Polymorphism by an Allele-specific PCR Assay (AS-PCR)

In order to type the G/T polymorphism efficiently, an allele-specificPCR assay using BIOLASE Diamond DNA Polymerase from Bioline wasdeveloped. This polymerase requires a perfect match of the 3′ end of theprimer to the template, and a mismatch at this position will result inno (or very weak) amplification. In this way, it is possible todistinguish between wildtype, carrier or sick animals by identifying thepresence or absence of allele-specific PCR products (FIG. 7). The leftpart of FIG. 7 shows the Allele-Specific PCR products of the codingstrand. As expected, wild type animals show amplification with theG-specific primer but not with the T-specific primer. The carriers showamplification of both the G- and T-specific primers, while sick animalsonly show amplification of the T-specific primer. The right part showsthe Allele-Specific PCR products of the non-coding strand, and asexpected, the patterns are the same as the coding strand. Wild typeanimals are homozygotic C, carriers are heterozygotic C/A, and sickanimals are homozygotic A.

From the above described results of using a positional candidate geneapproach, a bovine gene was identified which is homologous to the humangene SLC35A3 encoding a UDP-N-acetylglucosamine transporter. Within thisgene a G/T polymorphism was identified which alters the amino acidsequence of the protein. All affected calves analysed (39) arehomozygous for the T-allele (T/T) and known carriers (108) are allheterozygous for the polymorphism (G/T). Analysis of more than 500animals of the Holstein breed, chosen as being unrelated to Bell, failedto identify any animals carrying the T-allele. More than 1500 animalswere analysed having Bell in the pedigree without finding any unaffectedanimal being homozygous for the T-allele. Furthermore, more than 300cattle selected from 12 different breeds were analysed without detectingthe T-allele in any of these animals. Taken together, the findingsdescribed in the present application demonstrate that the T-allele ispresent in a single copy in animals which are carriers of CVM and in twocopies in animals affected by CVM. Detection of the T-allele in position559 (numbering from FIG. 4) is therefore diagnostic for CVM and idealfor detection of carriers of the CVM defect.

As the G/T polymorphism has been identified as being causative for CMV,any genetic markers closely coupled to this polymorphism may bediagnostic for CMV in a bovine population. Accordingly, the presentinvention describes a method to identify bovine subjects either affectedby CMV or carriers of CMV by determining the presence of the G/Tpolymorphism at position 559 of the bovine SLC35A3 gene, eitherindirectly by analysing any genetic markers, such as microsatellitesdescribed herein, coupled to the bovine SLC35A3 gene or directly byanalysing the sequence of the bovine SLC35A3 gene, e.g. as describedabove and in further details in the examples.

Within the scope of the present invention is therefore a method fordetecting and/or quantifying the presence of a genetic marker associatedwith bovine CVM in a bovine subject in order to be able to identify theCMV affected bovine subjects or carriers of CMV. The steps of the methodcomprises:

-   -   a) providing a bovine genetic material, and    -   b) detecting, in said genetic material, the presence or absence        of at least one genetic marker that is linked to a bovine        complex vertebral malformation disease trait.

The at least one genetic marker is linked to a gene causing bovine CMVdisease, said gene being identified herein to be the bovine SLC35A3 genewhich encodes the bovine SLC35A3 protein comprising an amino acidsequence as shown in SEQ ID NO: 17.

More specifically, the present invention relates to a method fordetecting bovine CMV, wherein the genetic marker is a single nucleotidepolymorphism at a position equivalent to nucleotide 559 of SEQ ID NO:18,said single nucleotide polymorphism being a G/T polymorphism.

The present application furthermore describes an efficient assay for thegenotyping of the present polymorphism using allele-specific PCR. Thisis but one of a battery of methods for the typing of SNPs, other methodswhich could be employed include, but are not limited to,mini-sequencing, primer-extension, pyro-sequencing, PCR-RFLP,allele-specific rolling circle-amplification, primer-extension followedby MALDI-TOF mass-spectrometry as well as a range of other enzymatic andhybridisation-based methods.

A phenotype resembling CVM has been demonstrated to exist in micemutated in the gene lunatic fringe (Evrad et al., 1998). Similar tocalves affected by CVM, mice homozygous for a null mutation in lunaticfringe exhibit an altered somite segmentation and patterning, having ashortened body axis, vertebral- and rib-fusions and incompletely formedvertebrae (Evrad et al., 1998). Fringe seems to participate in thedefinition of boundary formation and somite patterning by modulating theactivity of notch receptors (Klein and Arias, 1998; Moloney et al.,2000, Brückner et al., 2000). The Notch-modulating activity seems to bemediated by an N-acetylglucosaminyl-transferase activity of Fringe,which in Golgi initiates the elongation of O-linked fucose residuesattached to EGF-like sequence repeats of Notch (Moloney et al., 2000;Brückner et al., 2000).

Furthermore, as the bovine SLC35A3 gene is homologous to the humanSLC35A3 gene, it is, with the information given herein, obvious toanalyse the coding sequence of the human SLC35A3 gene for causative anddiagnostic mutations when studying human developmental defects,especially involving somite-segmentation and patterning.

The effect of a mutation in the Golgi-located N-acetylglucosaminyltransporter (SLC35A3) affecting transport of N-acetylglucosamine fromthe cytosol into the lumen of Golgi would be expected to deprive theFringe family of proteins for their substrate. This would affect theability of Fringe to modulate Notch activity and thereby cause asegmentation defect like CVM. It therefore seems very plausible that themutation in SLC35A3, apart from being diagnostic for CVM, is also themutation causing this widespread genetic defect.

The invention is described in further details in the following examples:

EXAMPLES Example 1

Genetic Mapping of Complex Vertebral Malformation (CVM)

This example illustrates the localisation of the CVM gene locus tobovine chromosome BTA3. Additionally, this embodiment describes theidentification of markers linked to the CVM gene locus, and thus thecharacterisation of the CVM region of bovine chromosome BTA3.

In order to map the locus responsible for CVM, samples were obtainedfrom animals participating in a breeding study. Briefly, approx. 300cows and heifers descending from the bull T Burma and inseminated withsemen from the bull KOL Nixon were selected for the breeding study.Thirteen affected calves were selected on the basis of the post mortemexamination, as described in Agerholm et al., 2000. These 13 calves aswell as their parents, in total 28 animals, were used in the initialgenome scan. The calves were separated by 4 generations to their commonancestor, the purebred bull Carlin-M Ivanhoe Bell.

The genome scan was conducted, covering all 29 autosomes, using abattery of micro-satellite markers picked from the USDA genome map(Kappes et al., 1997). Markers were selected with pair-wise distancesbetween 10 and 20 cM. In areas of doubt due to low marker informativity,new markers were included and typed. A total of 194 markers were used.PCR reactions were performed in duplexes in a volume of 5 μl in an ABI877 PCR robot (Applied Biosystems), containing 12 ng of genomic DNA,1×PCR buffer, 0.4 U AmpliTaq Gold (Applied Biosystems), 20 mmol of eachprimer and 2.0 mM MgCl₂. All markers were run at the same touchdown PCRconditions: Incubation at 94° C. for 12 minutes to activate the enzyme,35 cycles at 94° C., 30 sec; Ta, 45 sec; 72° C., 20 sec, ending with afinal extension at 72° C. for 10 min. The first ten cycles Ta decreasedfrom 67° C. to 58° C., one degree for each cycle, and the remaining 25cycles Ta were fixed at 58° C. PCR products were pooled and 5 to 9different markers were run in each lane on an ABI 377 (AppliedBiosystems), and gels were analysed with the accompanying software.Alleles were assigned with the Genotyper programme (Version 2.1, AppliedBiosystems).

For three markers, two-point lod scores were calculated using the MLINKprogramme of the LINKAGE package (Lathrop et al., 1985). Due to thepedigree structure (FIG. 1) with multiple inbreeding loops, the pedigreewas divided into thirteen small families, one for each affected calfincluding the Sire (KOL Nixon), the dam and the maternal grandsire (TBurma). The disease was assumed to be recessively inherited with acomplete penetrance of the genotype.

Significant linkage was found for all three markers. The highest lodscore (Z) was observed with BMS2790 and ILSTS029 with Z=10.35 at θ=0.Furthermore, the nearby marker INRA003 was also significantly linked tothe CVM locus (Table 3).

TABLE 3 Two point lod scores (Z) and recombination fractions (θ) fromthe linkage analysis of the CVM locus and bovine chromosome 3 markers.Recombination fraction Lod score Marker (θ) (Z) BMS2790 0.00 10.35INRA003 0.03 6.44 ILSTS029 0.00 10.35

The above results locate the CVM locus to BTA3 (FIG. 2) according to theUSDA genetic map (Kappes et al., 1997).

-   -   Eleven calves were homozygous for the interval defined by        INRA003, BMS2790, ILSTS029, BMS862 and HUJ246, while BMS2790 and        ILSTS029 alone were homozygous in all thirteen calves as        depicted in FIG. 1. It was possible to construct haplotypes of        these markers, allowing us to deduce the most likely CVM        haplotype (FIG. 1). The haplotypes are defined by the size of        the marker alleles which are numbered from 1 to N where 1        defines the shortest allele of the amplified marker and N        defines the longest allele. The actual length of the alleles        associated with CVM in Bell is as follows:

INRA003 (allele no. 3): 176 base pairs

BMS2790 (allele no. 3): 118 base pairs

ILSTS029 (allele no. 2): 164 base pairs

BMS862 (allele no. 1): 130 base pairs

HUJ246 (allele no. 3): 262 base pairs

The actual length of the alleles will depend upon the primers used toamplilfy the marker, and the fragment lengths shown above is based onusing the primers described in Table 4.

Furthermore, seventeen additional affected calves sampled as part of theDanish surveillance programme for genetic defects were included in thestudy. All affected animals had the pure-bred bull Bell as a commonancestor. DNA was extracted from blood samples or semen using standardprocedures.

The 17 calves and their mothers were genotyped with the 8 markersINRA003, BMS2790, ILSTS029, BM220, INRA123, BMS862, BMS937, and HUJ246spanning the region on BTA3 from approximately 59.5 cM to 67.9 cM, andsince the CVM gene in the additional 17 calves, like in the initialbreeding study, was assumed to originate from the common ancestor bullBell, a similar region of identity by descent (IBD) was expected toexist in all affected calves. This also turned out to be the case: in 17out of 17 calves the chromosome segment defined by INRA003 and BMS2790was homozygous, sharing the same alleles as the animals from thebreeding study. In two of the nineteen animals, heterozygosity wasobserved in the ILSTS029 and BMS862 locus explained by singlerecombination events between BMS2790 and ILSTS029. Thus, based on thecombined genotyping results, we have found that the CVM genetic defectis most likely located in an interval of less than 6 cM, flanked by themarkers INRA003 and ILSTS029 as illustrated in FIG. 2 and denoted “CVMregion”.

The sequences of the primers for the applied 8 markers INRA003, BMS2790,ILSTS029, BM220, INRA123, BMS862, BMS937, and HUJ246, are depicted inTable 4 below.

TABLE 4 Genetic SEQ ID marker Sequence of primers NO INRA003 F: CTG GAGGTG TGT GAG CCC CAT TTA SEQ ID NO: 1 R: CTA AGA GTC GAA GGT GTG ACT AGGSEQ ID NO: 2 BMS2790 F: AAG ACA AGG ACT TTC AGC CC SEQ ID NO: 3 R: AAAGAG TCG GAC ATT ACT GAG C SEQ ID NO: 4 ILSTS029 F: TGT TTT GAT GGA ACACAG CC SEQ ID NO: 5 R: TGG ATT TAG ACC AGG GTT GG SEQ ID NO: 6 INRA123F: TCT AGA GGA TCC CCG CTG AC SEQ ID NO: 7 R: AGA GAG CAA CTC CAC TGT GCSEQ ID NO: 8 BM220 F: TTT TCT ACT GCC CAA CAA AGT G SEQ ID NO: 9 R: TAGGTA CCA TAG CCT AGC CAA G SEQ ID NO: 10 HUJ246 F: ACT CCA GTT TTC TTTCCT GGG SEQ ID NO: 11 R: TGC CAT GTA GTA GCT GTG TGC SEQ ID NO: 12BMS862 F: TAT AAT GCC CTC TAG ATC CAC TCA SEQ ID NO: 13 R: ATG GAA AAATAA GAT GTG GTA TGT SEQ ID NO: 14 G BMS937 F: GTA GCC ATG GAG ACT GGACTG SEQ ID NO: 15 R: CAT TAT CCC CTG TCA CAC ACC SEQ ID NO: 16

Example 2

Diagnostic Test to Identify CVM Carriers:

A diagnostic test to determine bovine carriers of CVM was established bydetermining whether descendants from Bell were carriers of thedisease-associated haplotype.

The test was based upon the 8 microsatellite markers INRA003, BMS2790,ILSTS029, BM220, INRA123, BMS862, BMS937 and HUJ246, and relied upon therecognition of the disease specific alleles or haplotype (see FIG. 1) inanimals descending from Bell.

Animals were thus determined to be carriers if they had inherited thedisease-associated alleles in the region defined by the markers INRA003,BMS2790, ILSTS029, and BM220 from Bell or from animals descending fromBell. If the animals had not inherited the disease-associated haplotypefrom Bell or from animals descending from Bell, they were determined tobe non-carriers. In cases where the Bell haplotype had been split byrecombination, the animals were designated as indeterminable. The fouradditional markers were only used when the information content in thetest markers was decreased due to inability to distinguish betweenmaternal and paternal inheritance.

Like all diagnostic genetic tests based upon linked DNA markers, the CVMtest suffers from the drawback that a double recombination event (oneevent at each side of the causative gene, between the gene and theflanking markers) cannot be detected. In the present case, this eventwill be extremely rare due to the tight linkage between the markers andthe CVM gene, and the reliability of the test is estimated to be higherthan 99%.

Example 3

Tissue Dissection and RNA Isolation and cDNA Synthesis:

Roughly 5 grams of heart tissue were dissected from dead-born calveswithin 3 hours of delivery, immediately frozen in liquid nitrogen andstored at −80° C. 250 mg of tissue was used for RNA isolation. RNA wasisolated using the RNA Isolation Kit from Stratagene (cat. 200345).

cDNA was synthesised by mixing 2.5 μg of total RNA with 1 μl of oligo(dT)₁₂₋₁₈ (500 μg/ml), 1 μl of 10 mM dNTP mix and H₂O to give a finalvolume of 12 μl . The resulting mixture was heated at 65° C. for 5 min,chilled on ice and spun briefly. Following the addition of 4 μl of 5×first-strand buffer, 2 μl of 0.1 M DTT and 1 μl of H₂O, the contentswere mixed and incubated at 42° C. for 2 min, after which 1 μl (200 U)of Superscript II (GibcoBRL® Lifetechnologies) was added and theincubation allowed to continue at 42° C. for 1.5 hours. The reaction wasinactivated at 70° C. for 15 min. To remove RNA, the cDNA was incubatedat 37° C. for 20 min with 1 U RNase H (Roche Molecular Biochemicals).

Example 4

Sequencing of SLC35A3:

Based on a homology alignment of the SLC35A3 gene between homo sapiensand canis familiaris, 2 oligos (SL1F and SL8R) were designed foramplification of almost the entire cDNA for bovine SLC35A3, includingthe start codon. To obtain the 3′ end of the gene, the resulting PCRfragment was sequenced and a new oligo designed (SL5F). To amplify the3′ end of SLC35A3, SL5F was used in combination with an oligo(bSLCBVIR), based on a published sequence of a bovine EST.

The same PCR conditions were applied for both primer sets.

The PCR reactions were performed in a GeneAmp® PCR System 9700 (AppliedBiosystems) in a final volume of 10 μl consisting of 1 μl of 10×NH₄reaction buffer, 0.5 μl of 50 mM MgCl₂, 0.8 μl of dNTPs (2.5 mM ofeach), 5.65 μl H₂O, 1 μl of forward and reverse primer (5 pmol of each)and 0.05 μl of 5 U/μl BIOTAQ DNA polymerase (Bioline).

The touchdown PCR reaction consisted of an initial heat activation stepat 95° C. for 2 min followed by 10 cycles of denaturation for 30 sec at95° C., annealing at 62° C. for 30 sec (0.5° C. decrements), andelongation for 20 sec at 72° C., plus an additional 30 cycles with adenaturation step for 30 sec at 95° C., an annealing temperature of 57°C. for 30 sec and an elongation step at 72° C. for 30 sec.

The following primers were used to amplify the complete SLC35A3 cDNA:

SL1F: 5′-GGA GGC AAA TGA AGA TAA AAC-3′ (SEQ ID NO: 19) SL8R: 5′-CTA TGCTTT AGT GGG ATT3-′ (SEQ ID NO: 20) SL5F: 5′-GAG TTG CTT TTG TAC AGTGG-3′ (SEQ ID NO: 21) bSLCBVIR: 5′-ACT GGC TAC TAT CTA GCA CAG GA-3′(SEQ ID NO: 22)

The complete cDNA sequence was obtained by applying these primers infour separate cycle sequencing reactions using purified PCR products asthe template. The PCR products were purified using SPIN-X® (CorningIncorporated) from a 0.8% Seakem agarose gel.

Cycle sequencing reactions were carried out in a GeneAmp® PCR System9700 (Applied Biosystems) and included an initial step at 96° C. for 2min followed by 99 cycles of 96° C. for 10 sec, 55° C. for 5 sec and 60°C. for 4 min. Sequencing products were precipitated with two volumes ofethanol and 1/10 volume of 3 M NaAc (pH 5.5), washed with 70% ethanol,resuspended in 5 μl of loading buffer and run on 4% acrylamidesequencing gels using an ABI377 automatic sequencer.

The cDNA sequence of the bovine SLC35A3 is shown in FIG. 4 (SEQ ID NO:18).

Example 5

Identification and Isolation of BACs Containing Microsatellite Markers

Filter Hybridisation:

The filters were pre-hybridised in hybridisation solution (6×SSC (52.6 gof NaCl, 26.46 g of sodium citrate per liter), 5× Denhardt (2 g officoll (type 400, Pharmacia), 5 g of polyvinylpyrrolidone, 5 g of bovineserum albumin (Fraction V, Sigma), 0.5% SDS and 50 μg/ml SS-DNA) at 65°C. for 3 hours with rotation. 100 μl of 5′-end labelled oligonucleotidewas then incubated with the filters for 16 hours at 65° C. For endlabelling, 5 pmol of oligonucleotide was combined with 5 μl (50 μCi) ofgamma-³²P-ATP (specific activity>5000 Ci/mmole), 2 μl of 10× kinasebuffer, 11 μl of H₂O and 1 μl (10 U) of T4 polynucleotide kinase (NewEngland Biolabs Inc.), and the mix was incubated at 37° C. for 1.5 hoursfollowed by 5 min of boiling to heat inactivate the enzyme. The labelledprobe was NaAc/ethanol precipitated using standard procedures, and aftera wash in 70% ethanol, the probe was redissolved in 100 μl of H₂O.Following hybridisation the filters were washed once with wash solutionI (2×SSC, 0.2% SDS) and twice at 65° C. with wash solution II (0.1×SSC,0.5% SDS), and exposed to Kodak BIOMAX™ MS film for 2 days at −80° C.

The following 5′-end labelled oligonucleotides were used to identifyILSTS029 and INRA003 positive BAC dones:

ILSTS029 oligo: 5′-CAC ACC GCT GTA CAG GAA AAA GTG TGC CAA CCC TGG TCTAAA (SEQ ID NO: 23) TCC AAA ATC CAT TAT CTT CCA AGT ACA T-3′ INRA003oligo: 5′-CGT CCC CTA TGC GCT TAC TAC ATA CAC TCA AAT GGA AAT GGG (SEQID NO: 24) AAA ACT GGA GGT GTG TGA GCC CCA TTT A-3′PCR Screening of the Bovine BAC Library:

BAC pools were prepared from the BAC library and screened by PCR. ThePCR reactions were performed in a GeneAmp® PCR System 9700 (AppliedBiosystems) in a final volume of 10 μl consisting of 1 μl of 10×NH₄reaction buffer, 0.5 μl of 50 mM MgCl₂, 0.8 μl of dNTPs (2.5 mM ofeach), 5.65 μl of H₂O, 1 μl of forward and reverse primer (5 pmol ofeach) and 0.05 μl of 5 U/μl BIOTAQ DNA polymerase (Bioline).

The following primers were used to identify BMS2790 containing BACclones by PCR:

BMS2790F: 5′-AAG ACA AGG ACT TTC AGC CC-3′ (SEQ ID NO: 25) BMS2790R:5′-AAA GAG TCG GAC ATT ACT GAG C-3′ (SEQ ID NO: 26)

The touchdown PCR reaction consisted of an initial heat activation stepat 95° C. for 2 min followed by 10 cycles of denaturation for 30 sec at95° C., annealing at 70° C. for 30 sec (0.5° C. decrements), andelongation for 20 sec at 72° C., plus an additional 30 cycles with adenaturation step at 95° C. for 30 sec, annealing at 65° C. for 30 secand elongation at 72° C. for 20 sec.

BAC DNA Isolation and Sequencing:

BAC DNA was prepared according to Qiagens Large Construct Kit, andapproximately 1 μg of BAC DNA was used as the template for cyclesequencing performed with the BigDye™ Terminator Cycle Sequencing Kit(PE Applied Biosystems). The cycle sequencing reactions were performedin a final volume of 6 μl containing 1 μl of Big Dye™ Terminator mix, 1μl of primer (5 pmol), 1 μl of reaction buffer and 2 μl of H₂O. Cyclesequencing reactions were carried out in a GeneAmp® PCR System 9700(Applied Biosystems) and included an initial step at 96° C. for 2 minfollowed by 130 cycles of 96° C. for 10 sec, 55° C. for 5 sec and 60° C.for 4 min. Sequencing products were precipitated with two volumes ofethanol and 1/10 volume of 3 M NaAc (pH 5.5), washed with 70% ethanol,resuspended in 2 μl of loading buffer and run on 4% acrylamidesequencing gels using an ABI377 automatic sequencer.

Sequencing Primers:

T7: 5′-TTA TAC GAC TCA CTA TAG GG-3′ (SEQ ID NO: 27) SP6: 5′-ATT TAG GTGACA CTA TAG-3′ (SEQ ID NO: 28) INRA003F: 5′-CTG GAG GTG TGT GAG CCC CATTTA-3′ (SEQ ID NO: 29) INRA003R: 5′-CTA AGA GTC GAA GGT GTG ACT AGG-3′(SEQ ID NO: 30)

Example 6

Determination of CVM Status by Sequencing:

PCR reactions (2 μl of purified template/sample genomic DNA, 2 μl of10×PCR buffer, 2 μl of 25 mM MgCl₂, 3.3 μl of 0.2 mM of each dNTP(Ultrapure dNTP, 27-2033-01; Amersham Pharmacia Biotech), 6 pmol ofprimer (forward: CBFEX1, 5′-GGC CCT CAG ATT CTC-3′ (SEQ ID NO: 31);reverse: CBTEXR, 5′-GTT GAA TGT TTC TTA-3′) (SEQ ID NO: 32), 0.165 U Taqpolymerase (Biotaq, M95801B; Bioline), dH₂O ad total volume) wereperformed oil-free in 96-well plates using a Primus HT (MWG Biotech AG).Cycling conditions: 95° C. 120 sec, 35×[95° C. 60 sec, 60° C. 30 sec,72° C. 110 sec]. Post-reaction clean-up was done by gel filtration(Millipore Filtration System) with Sephadex G-50 Superfine (17-0041-01,Amersham Pharmacia Biotech) carried out according to the manufacturer'srecommendation, using 50 μl of dH₂O for final sample elution. Forwardand reverse sequencing reactions were performed with the same primers asused for the generation of the PCR product (2 μl of PCR product, 8 μl ofSequencing Mix, 0.6 μl of 6 pmol Primer (see above), dH₂O ad 20 μl;DYEnamic ET Dye Terminator Cycle Sequencing Kit (US81095). Afterthermocycling (30×[95° C. 20 sec, 55° C. 15 sec, 60° C. 70 sec]) sampleswere cleaned by gel filtration essentially as described above andanalysed on a MegaBACE1000 (Amersham Pharmacia Biotech) using LPAlong-read matrix and the following sequencing conditions: 90 secinjection at 3 kV and 35 min run time at 9 kV.

Example 7

Allele-specific PCR Assay:

Primers were designed from the cDNA sequence and the fourallele-specific primers were designed to have the 3′ base at theposition of the mutation.

Primer sequences:

T_fwd: 5′-CAG TGG CCC TCA GAT TCT CAA GAG CTT AAT TCT AAG GAA CTT TCA(SEQ ID NO: 33) GCT GGC TCA CAA TTT GTA GGT CTC ATG GCA T-3′ G_fwd:5′-CAC AAT TTG TAG GTC TCA TGG CAG-3′ (SEQ ID NO: 34) A_rev: 5′-GCC ACTGGA AAA ACA TGC TGT GAG AAA-3′ (SEQ ID NO: 35) C_rev_link*: 5′-aat gctact act att agt aga att gat gcc acc ttt tca gct cgc gcc cca aat (SEQ IDNO: 36) gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta tct aatggt caa act ttt ttC TGG AAA AAC ATG CTG TGA GAA C-3′ Fwd: 5′-GGC CCT CAGATT CTC AAG AGC-3′ (SEQ ID NO: 37) Rev: 5′-CGA TGA AAA AGG AAC CAA AAGGG-3′ (SEQ ID NO: 38) The C_rev_link primer contains a linker sequencefrom the M13 phage (shown in lower case letters). This linker was addedto obtain a longer PCR product in order to be able to multiplex the C-and A-primers in one PCR reaction. The 3′ base at the position of themutation is shown in bold. The C- and G-primers are specific for thewildtype allele, while the T- and A-specific primers are specific forthe mutation.

Primer pairs:

C- and A-specific multiplex: Fwd+A_rev+C_rev_link (lower strand)

T-specific reaction: T_fwd+Rev (upper strand)

G-specific reaction: G_fwd+Rev (upper strand)

AS-PCR Conditions:

Each PCR reaction was carried out in a 10 μl volume containing 20–100 ngof genomic DNA, 0.025 units/μl of BIOLASE Diamond DNA polymerase, 0.75mM dNTPs, 3 mM MgCl₂, 0.25 pmol/μl primer (0.125 pmol/μl of the tworeverse primers in the multiplex) in 1×NH4 buffer (Bioline). PCR wascarried out in a GeneAmp® PCR System 9700 (PE Applied Biosystems) underthe following conditions: 95° C. for 4 min, 35 cycles of 94° C. for 30s, 62° C. (56° C. for the T- and G-reaction) at ramp 80% for 30 s, and72° C. for 30 s followed by a final extension at 72° C. for 7 min andstorage at 4° C. PCR was followed by electrophoresis in a 2% agarose gelat 200 V for 30 min.

The results of the allele-specific PCR analysis of two wildtype, twocarriers, and two sick animals are shown in FIG. 7.

Figure Legends

FIG. 1 shows the pedigree used to locate the bovine complex vertebralmalformation (CVM) locus and haplotypes of five microsatellite markerson bovine chromosome 3. The most likely CVM haplotypes are in bold.Filled black squares represent affected calves. Double lines between thesire and the dams indicate inbreeding loop. N refers to the number ofanimals. Genotypes of the thirteen different dams are for simplicityreasons not shown.

FIG. 2 shows the genetic map of bovine chromosome 3. Numbers on thesides refer to the genetic distances given in centiMorgan (cM) along thechromosome. The most likely location of the bovine complex vertebralmalformation (CVM) locus is indicated.

FIG. 3 shows the relative distance in cM between the 3 microsatellitemarkers ILSTS029, BMS2790 and INRA003 (shown on the line denoted Contigmarkers on bovine Chr. 3) on the bovine chromosome 3 as depicted by theU.S. Meat Animal Research Center (Kappes et al. 1997). BACs containingthese 3 markers were isolated either by hybridisation to high densityreplica filters (ILSTS029 and INRA003), or by PCR screening of theRPCI-42 bovine BAC library (BMS2790). The identified BACs are shown inblack bars and annotated by plate number/well number. These BACs weresubjected to end-sequencing using SP6 and T7 primers or to sequencingusing primers extending from the microsatellite. The resulting sequenceswere blasted against the human chromosome 1 using the Ensemble Server atthe Sanger Centre. The accession numbers from the blast search are shownas numbers under the human chromosome 1 and the relative distancebetween the hits is given in MB. Selected genes in the region are shownin boxes.

FIG. 4 shows the cDNA sequence and translation of the SLC35A3 gene (SEQID NO: 18) and the encoded amino add sequence (SEQ ID NO: 17). Thepolymorphic nucleotide in position 559 and the affected valine-180 isindicated in bold.

FIG. 5 shows a comparison of the deduced amino add sequence of cowSLC35A3 with human (AB021981) (Ishida et al. 1999) and dog (AF057365)(Guillen et al. 1998) sequences. Dots indicate residues that match theBos Taurus sequence. Dashes indicate gaps that have been introduced tooptimise the alignment.

FIG. 6 shows the results obtained from sequencing the region (fromnucleotide 544 to 572 of SLC35A3, see FIG. 4) showing the G/Tpolymorphism in position 559 in determination of CVM status bysequencing. The left and right panels show forward and reversesequencing, respectively. The upper row (−/−) shows the sequencing of awildtype animal, the middle row shows the sequencing of a carrier(heterozygote), and the lower row shows the sequencing of an affectedanimal.

FIG. 7 is a picture showing the Allele-Specific PCR products from twowildtype, two carriers, and two sick animals. Annotations: WT: wildtype,C: carrier, S: sick, neg: negative control, M: marker (size ladder).Arrows show the allele-specific PCR products: C: 220 bp, A: 98 bp, T:340 bp, and G: 288 bp.

REFERENCES

-   1. Agerholm J S, Bendixen, C., Andersen O., Arnbjerg, J. (2000) LK    meddelelser October 2000.-   2. Barendse W, Valman D, Kemp S J, Sugimoto Y, Armitage S M,    Williams J L, Sun H S, Eggen A, Agaba M, Aleyasin S A, Band M,    Bishop M D, Bultkamp J, Byrne K, Collins F, Cooper L, Coppettiers W,    Denys B, Drinkwater R D, Easterday K, Elduque C, Ennis S, Erhardt G,    Li L, Lil L (1997) A medium-density genetic linkage map of the    bovine genome. Mamm Genome 8, 21–28.-   3. Brückner, K., Perez, L., Clausen, H., & Cohen, S., (2000)    Glycosyltransferase activity of Fringe modulates Notch-Delta    interactions Nature 406, pp. 411–415.-   4. Evrad, Y. A., Lun, Y., Aulehla, A,. Gan, L, &    Johnson, R. L. (1998) Lunatic fringe is an essential mediator of    somite segmentation and patterning. Nature 394, pp. 377–381.-   5. Guillen, E., Abeijon, C., & Hirschberg, C. B. (1998) Mammalian    Golgi apparatus UDP-N-acetylglucosamine transporter: Molecular    cloning by phenotypic correction of a yeast mutant. Proc. Natl.    Acad. Sci. USA. 95, pp. 7888–7892.-   6. Ishida, N., Yoshioka, S., Chiba, Y., Takeuchi, M., &    Kawakita, M. (1999) Molecular cloning and functional expression of    the human golgi UDP-N-acetylglucosamine transporter. J. Biochem.    126, pp. 68–77.-   7. Kappes S M, Keele J W, Stone R T, McGraw R A, Sonstegard T S,    Smith T P, Lopez-Corrales N L, Beattie C W (1997) A    second-generation linkage map of the bovine genome. Genome Res 7,    235–249.-   8. Klein, T., & Arias, M. (1998) Interactions among Delta, Serrate    and Fringe modulate Notch activity during drosophila wing    development. Development 125, pp. 2951–2962.-   9. Lathrop G M, Lalouel J M, Julier C, Ott J (1985) Multilocus    linkage analysis in humans: detection of linkage and estimation of    recombination. Am J Hum Genet 37, 482–498-   10. Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao,    L, Wilson, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S., &    Vogt, T. F. (2000) Fringe is a glycosyltransferase that modifies    Notch. Nature 406, pp 369–375.-   11. Solinas-Toldo S, Lengauer C, Fries R (1995) Comparative genome    map of human and cattle Genomics 27, 489–496.

1. A method for detecting bovine complex vertebral malformation (CVM) ina Holstein-Friesian subject said method comprising a) providing aHolstein-Friesian genetic material comprising a coding sequence whichencodes either i) SEQ ID NO:17; or ii) a polypeptide which differs fromSEQ ID NO:17 solely in that there is a phenylalanine at position 180,and b) detecting, in the Holstein-Friesian subject genetic material, thepresence or absence of complex vertebral malformation disease, whereinthe presence of a coding sequence encoding polypeptide ii) is indicativeof CVM.
 2. The a method according to claim 1, wherein the detection ofthe presence or absence of the coding sequence encoding polypeptide (ii)is performed by a technique selected from the group consisting ofallele-specific PCR, minisequencing, primer-extension, pyro-sequencing,PCR-RFLP, allele-specific rolling circle amplification andprimer-extension followed by MALDI-TOF mass-spectrometry.